The discovery that restricted regions of the adult mammalian brain exhibit ongoing neurogenesis promises considerable therapeutic potential, however, there is a current lack of understanding regarding the cellular and molecular mechanisms that control neural stein cell proliferation and fate specification in vivo. Neuronal differentiation is in part controlled by the transcription factor NRSF, which restricts expression of neuron specific genes to neuronal cells. By binding to a ~21 base-pair consensus DNA sequence (NRSE) within their regulatory regions and via recruitment of silencers, NRSF represses these genes. As neural stem cells (NSCs) undergo differentiation into mature neurons, repression is relieved and expression of neuron-specific genes is activated. Recently, a double-stranded RNA (dsRNA) of the same sequence and approximate length as the NRSE was found to mediate this switch. This RNA is necessary and sufficient to specify the neuronal fate of multipotent adult NSCs in vitro and may therefore function as an intrinsic inducer of neuronal differentiation in vivo. Moreover, this finding represents the first example of a novel class of regulatory RNAs, termed small modulatory RNAs (smRNA), whose biogenesis and mode of action is distinct from previously characterized RNAs. The overall goal of this work is to elucidate how NRSE smRNA is expressed and to understand the molecular mechanism by which this RNA regulates adult neurogenesis in vivo. This project is in response to the PAS-04-130 on Collaborative Research in Stem Cell Biology and reflects the collaboration of several scientific groups to address this novel discovery. Specific Aim 1 will focus on how the NRSE smRNA is regulated in NSCs and Specific Aim 2 will examine the functional consequences of regulating this smRNA in vivo. These are aims directly related to skills and experience in the Gage lab. Specific Aim 3 focuses on the structural relationships between the NRSE smRNA and NRSF, and with its DNA binding sequence. The Riek lab has skills and expertise in addressing these structural biology questions. Specific Aim 4 continues the search for new smRNAs by taking advantage of the expertise in ribozyme technology developed by Kuwabara in Japan and applied at the Salk for this project.